The purpose of this project is to elucidate the structure and mechanism of formation of higher order assemblies of enamel matrix proteins and their influence on mineralization and crystal organization in vitro. We propose that organized mineral stuctures are generated within organized super-assemblies of matrix proteins in cooperation with other macromolecules that guide crystal growth and shape. Our working hypothesis is that higher order assemblies of full-length amelogenin, in association with soluble acidic proteins (e.g., enamelin and ameloblasrin), regulate the nucleation, growth, shape, and arrangement of initial enamel mineral crystals. Under appropriate mineralizing conditions, such assemblies support the formation of parallel arrays of very thin ribbons of enamel mineral. The growth of these enamel ribbons in thickness and width is immediately inhibited by soluble hydrophobic enamel proteins (e.g. sparingly soluble amelogenins) which adsorb onto specific faces of the growing crystals. It is further hypothesized that the subsequent onset of growth of these mineral ribbons during tissue maturation is controlled by the degradation of these enamel protein inhibitors by specific enamel proteinases. To improve our understanding of how matrix proteins regulate mineralization in tissues like enamel, we propose to characterize specific enamel matrix components with respect to their ability to form higher-order assemblies that ultimately regulate organized mineralization. Specifically, we propose to characterized the ability of key enamel matrix proteins to bind to mineral surfaces (Aim 1), to regulate crystal shape and kinetics of crystal growth (Aim 2), and to induce calcium phosphate formation in vitro (Aim 3). Importanly, these findings;will be integrated with biophysical studies to determine the structure and mechanism of formation of proposed higher order assemblies of the full-length amelogenin (Aim 4) and the mechanism by which such assemblies regulate the formation of organized mineral structures, similar to that of dental enamel (Aim 5). Long term, such information should provide new insights for the development of bio-inspired materials and novel approaches for mineralized tissue repair and regeneration.